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pY RNA1-s2: a highly retina-enriched small RNA that selectively binds to Matrin 3 (Matr3).

Yamazaki F, Kim HH, Lau P, Hwang CK, Iuvone PM, Klein D, Clokie SJ - PLoS ONE (2014)

Bottom Line: In contrast, pY RNA1-s1 does not bind these proteins.Hypothetically, pY RNA1-S2 might act to modulate cellular function through this molecular mechanism.The retinal enrichment of pY RNA1-s2 provides reason to suspect that the pY RNA1-s2/Matr3 interaction could play a role in vision.

View Article: PubMed Central - PubMed

Affiliation: Section on Neuroendocrinology, Program in Developmental Endocrinology and Genetics, The Eunice Shriver Kennedy National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, United States of America.

ABSTRACT
The purpose of this study was to expand our knowledge of small RNAs, which are known to function within protein complexes to modulate the transcriptional output of the cell. Here we describe two previously unrecognized, small RNAs, termed pY RNA1-s1 and pY RNA1-s2 (processed Y RNA1-stem -1 and -2), thereby expanding the list of known small RNAs. pY RNA1-s1 and pY RNA1-s2 were discovered by RNA sequencing and found to be 20-fold more abundant in the retina than in 14 other rat tissues. Retinal expression of pY RNAs is highly conserved, including expression in the human retina, and occurs in all retinal cell layers. Mass spectrometric analysis of pY RNA1-S2 binding proteins in retina indicates that pY RNA1-s2 selectively binds the nuclear matrix protein Matrin 3 (Matr3) and to a lesser degree to hnrpul1 (heterogeneous nuclear ribonucleoprotein U-like protein). In contrast, pY RNA1-s1 does not bind these proteins. Accordingly, the molecular mechanism of action of pY RNA1-s2 is likely be through an action involving Matr3; this 95 kDa protein has two RNA recognition motifs (RRMs) and is implicated in transcription and RNA-editing. The high affinity binding of pY RNA1-s2 to Matr3 is strongly dependent on the sequence of the RNA and both RRMs of Matr3. Related studies also indicate that elements outside of the RRM region contribute to binding specificity and that phosphorylation enhances pY RNA-s2/Matr3 binding. These observations are of significance because they reveal that a previously unrecognized small RNA, pY RNA1-s2, binds selectively to Matr3. Hypothetically, pY RNA1-S2 might act to modulate cellular function through this molecular mechanism. The retinal enrichment of pY RNA1-s2 provides reason to suspect that the pY RNA1-s2/Matr3 interaction could play a role in vision.

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Cellular distribution of Y RNA1 and pY RNA1-s2.A. Retinal tissue was obtained from three animals, pooled and dissociated in lysis buffer with the addition of RNAse inhibitors, as described in “Materials and Methods”. These preparations were pooled and partitioned into nuclear and cytoplasmic fractions. Equal proportions of each fraction were subjected to SDS-PAGE and western blotted for Pax 6 (nuclear fraction control), 14-3-3 (cytoplasmic control) and β-Actin (cytoplasmic control). B. Northern blot of total RNA extracted from cytoplasmic and nuclear fractions as described above, separated on a denaturing 15% TBE-Urea PAGE gel, using a probe directed toward pY RNA1-s2, indicating the predominance of pY RNA1-s2 in the cytoplasm, but also detectable in the nuclear fraction. The panel indicated by “Y RNA1” shows the un-processed Y RNA and the panel labeled “pY RNA1-s2” shows the processed Y RNA1 that migrates at ∼27 nt. The blot was re-probed with a probe directed to U6 (as a control) indicating the majority is present in the nucleus.
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pone-0088217-g005: Cellular distribution of Y RNA1 and pY RNA1-s2.A. Retinal tissue was obtained from three animals, pooled and dissociated in lysis buffer with the addition of RNAse inhibitors, as described in “Materials and Methods”. These preparations were pooled and partitioned into nuclear and cytoplasmic fractions. Equal proportions of each fraction were subjected to SDS-PAGE and western blotted for Pax 6 (nuclear fraction control), 14-3-3 (cytoplasmic control) and β-Actin (cytoplasmic control). B. Northern blot of total RNA extracted from cytoplasmic and nuclear fractions as described above, separated on a denaturing 15% TBE-Urea PAGE gel, using a probe directed toward pY RNA1-s2, indicating the predominance of pY RNA1-s2 in the cytoplasm, but also detectable in the nuclear fraction. The panel indicated by “Y RNA1” shows the un-processed Y RNA and the panel labeled “pY RNA1-s2” shows the processed Y RNA1 that migrates at ∼27 nt. The blot was re-probed with a probe directed to U6 (as a control) indicating the majority is present in the nucleus.

Mentions: To determine the subcellular location of processed pY RNA1-s2, cytoplasmic and nuclear protein and RNA extracts were prepared from rat retina and pineal tissues (Figure 5). Pax6 and the small nucleolar RNA U6 were used as markers for the nuclear fraction (Figure 5A; upper panel, Figure 5B; lowest panel) and 14-3-3 and β-actin were used as markers for the cytoplasmic fraction (Figure 5A; lower panels, Figure 5B; upper panels). Northern blot analysis revealed that the un-processed and processed Y RNA1 is enriched in the cytoplasm and detectable at lower levels in the nucleus (Figure 5B).


pY RNA1-s2: a highly retina-enriched small RNA that selectively binds to Matrin 3 (Matr3).

Yamazaki F, Kim HH, Lau P, Hwang CK, Iuvone PM, Klein D, Clokie SJ - PLoS ONE (2014)

Cellular distribution of Y RNA1 and pY RNA1-s2.A. Retinal tissue was obtained from three animals, pooled and dissociated in lysis buffer with the addition of RNAse inhibitors, as described in “Materials and Methods”. These preparations were pooled and partitioned into nuclear and cytoplasmic fractions. Equal proportions of each fraction were subjected to SDS-PAGE and western blotted for Pax 6 (nuclear fraction control), 14-3-3 (cytoplasmic control) and β-Actin (cytoplasmic control). B. Northern blot of total RNA extracted from cytoplasmic and nuclear fractions as described above, separated on a denaturing 15% TBE-Urea PAGE gel, using a probe directed toward pY RNA1-s2, indicating the predominance of pY RNA1-s2 in the cytoplasm, but also detectable in the nuclear fraction. The panel indicated by “Y RNA1” shows the un-processed Y RNA and the panel labeled “pY RNA1-s2” shows the processed Y RNA1 that migrates at ∼27 nt. The blot was re-probed with a probe directed to U6 (as a control) indicating the majority is present in the nucleus.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3928194&req=5

pone-0088217-g005: Cellular distribution of Y RNA1 and pY RNA1-s2.A. Retinal tissue was obtained from three animals, pooled and dissociated in lysis buffer with the addition of RNAse inhibitors, as described in “Materials and Methods”. These preparations were pooled and partitioned into nuclear and cytoplasmic fractions. Equal proportions of each fraction were subjected to SDS-PAGE and western blotted for Pax 6 (nuclear fraction control), 14-3-3 (cytoplasmic control) and β-Actin (cytoplasmic control). B. Northern blot of total RNA extracted from cytoplasmic and nuclear fractions as described above, separated on a denaturing 15% TBE-Urea PAGE gel, using a probe directed toward pY RNA1-s2, indicating the predominance of pY RNA1-s2 in the cytoplasm, but also detectable in the nuclear fraction. The panel indicated by “Y RNA1” shows the un-processed Y RNA and the panel labeled “pY RNA1-s2” shows the processed Y RNA1 that migrates at ∼27 nt. The blot was re-probed with a probe directed to U6 (as a control) indicating the majority is present in the nucleus.
Mentions: To determine the subcellular location of processed pY RNA1-s2, cytoplasmic and nuclear protein and RNA extracts were prepared from rat retina and pineal tissues (Figure 5). Pax6 and the small nucleolar RNA U6 were used as markers for the nuclear fraction (Figure 5A; upper panel, Figure 5B; lowest panel) and 14-3-3 and β-actin were used as markers for the cytoplasmic fraction (Figure 5A; lower panels, Figure 5B; upper panels). Northern blot analysis revealed that the un-processed and processed Y RNA1 is enriched in the cytoplasm and detectable at lower levels in the nucleus (Figure 5B).

Bottom Line: In contrast, pY RNA1-s1 does not bind these proteins.Hypothetically, pY RNA1-S2 might act to modulate cellular function through this molecular mechanism.The retinal enrichment of pY RNA1-s2 provides reason to suspect that the pY RNA1-s2/Matr3 interaction could play a role in vision.

View Article: PubMed Central - PubMed

Affiliation: Section on Neuroendocrinology, Program in Developmental Endocrinology and Genetics, The Eunice Shriver Kennedy National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, United States of America.

ABSTRACT
The purpose of this study was to expand our knowledge of small RNAs, which are known to function within protein complexes to modulate the transcriptional output of the cell. Here we describe two previously unrecognized, small RNAs, termed pY RNA1-s1 and pY RNA1-s2 (processed Y RNA1-stem -1 and -2), thereby expanding the list of known small RNAs. pY RNA1-s1 and pY RNA1-s2 were discovered by RNA sequencing and found to be 20-fold more abundant in the retina than in 14 other rat tissues. Retinal expression of pY RNAs is highly conserved, including expression in the human retina, and occurs in all retinal cell layers. Mass spectrometric analysis of pY RNA1-S2 binding proteins in retina indicates that pY RNA1-s2 selectively binds the nuclear matrix protein Matrin 3 (Matr3) and to a lesser degree to hnrpul1 (heterogeneous nuclear ribonucleoprotein U-like protein). In contrast, pY RNA1-s1 does not bind these proteins. Accordingly, the molecular mechanism of action of pY RNA1-s2 is likely be through an action involving Matr3; this 95 kDa protein has two RNA recognition motifs (RRMs) and is implicated in transcription and RNA-editing. The high affinity binding of pY RNA1-s2 to Matr3 is strongly dependent on the sequence of the RNA and both RRMs of Matr3. Related studies also indicate that elements outside of the RRM region contribute to binding specificity and that phosphorylation enhances pY RNA-s2/Matr3 binding. These observations are of significance because they reveal that a previously unrecognized small RNA, pY RNA1-s2, binds selectively to Matr3. Hypothetically, pY RNA1-S2 might act to modulate cellular function through this molecular mechanism. The retinal enrichment of pY RNA1-s2 provides reason to suspect that the pY RNA1-s2/Matr3 interaction could play a role in vision.

Show MeSH
Related in: MedlinePlus